The present invention relates to tissue processing, and more particularly it relates to a rapid tissue processing method and apparatus that uses ohmic energy and microwave energy to heat the process solution and tissue sample during processing. The present invention also relates to a tissue processing method that employs the use of environmentally friendly green chemicals.
A tissue sample must be processed before it can be analyzed for diagnostic or testing purposes. This processing acts to halt the degradation of cellular structure, and to stabilize the cellular characteristics, and to sufficiently harden the tissue so that extremely fine segments may be cut therefrom for purposes of analysis. Tissue processing is well known in the art of histology.
There are four general steps performed when processing a tissue sample: fixation, dehydration, clearing, and impregnation (sometimes herein also referred to as infiltration). These steps are generally effectuated by submerging the tissue sample in different solutions to produce chemical reactions. It is important that tissue samples be processed in a quality and uniform manner so that the analytical results and diagnosis are consistent and accurate. Some of the types of analysis that can be performed on tissue samples after the sample has been processed include visual analysis, gel electrophoresis, immunohistochemical stains, flow cytometry, and other genetic analysis. Physicians diagnose a variety of ailments and diseases based upon these analyses.
Fixation initiates preservation of the tissue specimen by cross linking proteins and halting cellular degradation. Without chemical fixation, endogenous enzymes will catabolize and lyse the cell, and the cellular microanatomy will be altered. Traditionally, fixatives have included ketones, aldehydes, alcohols, acetic acid, heavy metals, chromic acid, picric acid, or osmium tetroxide. Indications that fixation was inadequate can include disassociation of tissue structures, bubbles in tissue sections, poor and irregular staining, shrunken cells, clumping of cytoplasm, condensation and less distinct nuclear chromatin, and autolysisihemolysis of erythrocytes. A failure to preserve the micro-anatomical structure of the cellular specimen may adversely impact the analysis and lead to the potential for misdiagnosis.
Dehydration removes water from the tissue specimen to promote hardening. Specifically, the water molecules that reside in the space between the membranes of the cells making up the specimen are evacuated and replaced with molecules of the dehydrating agent. Replacement of this water in the tissue specimen with a dehydrating agent also facilitates subsequent replacement of the dehydrating agent with the solidifying material used in impregnation. This solution exchange is enhanced by using a volatile solvent for dehydration. The dehydrating agent may be low molecular weight alcohols, ketones, dioxane, alkylene glycols, or polyethylene glycols. Failure to dehydrate the specimen can lead to inadequate impregnation, poor ribbon formation during sectioning, clefts in tissue sections, dissociation of structures, water crystals in tissue sections, and poor staining.
Clearing is a process that enhances the ability to visualize the internal characteristics of cellular structure. The clearing step includes extracting dehydrating agent from the tissue specimen to reduce the tissue's opacity. Examples of clearants have traditionally included xylene, limonene, benzene, toluene, chloroform, petroleum ether, carbon bisulfide, carbon tetrachloride, dioxane, clove oil, or cedar oil.
Defatting of the tissue routinely occurs during the clearing operation, and is the process of removing fat (lipids) from the tissue specimen. If left in place, the fatty molecules would impair clearing and impregnation. Specifically, inadequate fat removal can result in spreading artifacts of tissue sections, wrinkling of tissue sections, and poor staining.
Once the tissue specimen is suitably fixed, dehydrated, and cleared, it is hardened by impregnation with a support medium such as wax, celloidin, polyalkylene glycols, polyvinyl alcohols, agar, gelatin, nitrocelluloses, methacrylate resins, epoxy resins, or other plastics. This step involves migration of the hardening agent into tissue cavities and cells that make up the tissue specimen. Appropriate hardening of the tissue specimen with adequate preservation of cellular morphology is required prior to placing the impregnated specimen in a block and obtaining thin sections. Preferred impregnation materials are commercial wax formulae, mixtures of waxes of different melting points (e.g., liquid mineral oil and solid paraffin), paraplast, bioloid, embedol, plastics and the like. Paraffin is often preferred because it is inexpensive, easy to handle, and ribbon sectioning is facilitated by the coherence of structures provided by this material.
It has long been a goal to decrease tissue processing times, thus allowing quicker analysis and diagnosis of the tissue sample. This is particularly important when tissue analysis is required as a part of a surgical procedure. However, in decreasing tissue processing times, the integrity of the tissue sample must remain high to facilitate accurate diagnosis. Many of the attempts to reduce the time of tissue processing have included the addition of heat energy during the processing steps so as to speed up the migration of process solution throughout the specimen and to facilitate chemical reactions occurring within the respective steps. For example, U.S. Pat. Nos. 4,656,047, 4,839,194, and 5,244,787 disclose the use of microwave energy; U.S. Pat. Nos. 3,961,097 and 5,089,288 disclose the use of ultrasonic energy; and U.S. Pat. No. 5,023,187 disclose the use of infrared energy.
Many of these prior art methods are undesirable for a number of reasons. One reason is that cellular structures, especially DNA and RNA, degrade when exposed to heat for prolonged periods of time. Such degradation makes analysis and diagnosis difficult or even impossible, and increases the risks of a misdiagnosis.
Prior art tissue processing methods can be broken into two main groups: conventional processing and microwave heat processing. Conventional processing methods employ vacuum and pressure with the chemical processing steps discussed above. Conventional methods may or may not include the addition of heat energy; however, any heat used is not produced by microwave energy. Microwave processing methods employ heat, vacuum, and pressure wherein the heat is produced by microwave energy.
The problem with conventional (non-microwave) methods is three fold. First, conventional methods have traditionally used hazardous chemicals that are known carcinogens to effectuate the chemical reactions during processing. Second, all tissue types are lumped together during tissue processing so that both “bound water” and “free water” molecules are removed from the tissue sample during processing, which damages the tissue sample. The ultimate goal of processing is to infiltrate the specimen with a hardening agent that is not miscible with the fresh or fixed specimen. Tissue samples are filled with free water that simply occupies the spaces among the biological molecules of carbohydrates, proteins, lipids and other compounds. This free water must be removed if the hardening agent is to enter.
There is a second type of water molecule in tissue specimen. This is referred to as bound water. Bound water is found surrounding nearly all macromolecules, being held to their surfaces by weak hydrogen bonds. It is not free to move around inside the tissue. It is not necessary to remove bound water in order to infiltrate the specimen with a hardening agent. Removal of bound water causes excessive hardening and shrinkage of the tissue sample. Also, bound water acts as a spacer among adjacent macromolecules, keeping them from coming close enough together to form hydrogen bonds between themselves. Once this insulating layer of bound water is removed, the tissue shrinks as macromolecules concentrate, and the specimen changes its cutting consistency as these molecules bind together. Fibrous tissue becomes tough and specimens comprised mostly of cells become crumbly. In conventional tissue processing, in the removal of free water from the larger, dense segments of the tissue specimen, bound water is also inadvertently removed from smaller, less dense segments, thus degrading those segments of the specimen. Third, for conventional processing methods processing times are usually in excess of 10 hours which means patients often have to wait until the following day for the tissue processing, analysis, and diagnosis. This relatively long time period can be detrimental and even life threatening for patients in situations where tissue analysis is urgently required. Because of this extended length of time, conventional tissue processing techniques may not be used for surgical analysis.
One problem with prior art microwave methods is that the heating of the tissue sample lacks uniformity. As is known in microwave technology, microwave energy produces hot spots and cold spots within the material being heated (target material). See Leong, Anthony S-Y, Microwave Technology for Morphological Analysis, Cell Vision, Vol. 1, No. 4 (1994). With only moderate success, attempts to reduce the occurrence of hot spots and cold spots have included fans inside the microwave chamber comprised of special material to disseminate the microwave energy, and rotating platforms to try to provide the target material uniform exposure to the microwave energy. The temperature within the hot spots can cause tissue destruction. Within the cold spots, the temperature may be inadequate to trigger or to complete the required chemical reactions. The result of the hot and cold spots is a lack of consistent processing of all of the cellular tissue comprising the specimen. Such a lack of consistency may result in poor or improper staining or analysis. The resulting potential for misdiagnosis may be dangerous and even life threatening for patients.
Another problem with microwave methods is that microwave energy may affect the bound water that physically separates macromolecules. Microwave energy is converted to kinetic and chemical energy. The kinetic energy results from the rapid oscillation of dipolar molecules causing internal heat. Although microwaves cannot ionize molecules and are too small to break molecular bonds, use of excessive microwave energy can redistribute the hydrogen bonds (chemical energy). Excessive microwave exposure also causes thinning of the bound water layers. This allows bound water layers to unwind and form new crosslinks, resulting in the formation of different configurations with a resultant potential for misdiagnosis.
Another problem with microwave energy in tissue processing is that microwave energy is only capable of penetrating the tissue to only a limited depth. See Kok L P, Visser P E, Histoprocessing with the Microwave Oven: An Update, Histochem J. 1988 June-July; 20 (6-7): 323-8. This means that in order to effectively use microwave energy for tissue processing, the tissue samples must be very thin. Thin samples are more difficult to handle and they can also be more difficult to obtain. If the sample is not sufficiently thin, then the cells comprising the outer portion of the tissue sample may process differently than the cells comprising the inner portion of the tissue sample. Attempts to overcome this limitation by increasing the amount of microwave energy to penetrate more deeply into the sample can damage the cell structure, DNA, and RNA, as discussed above.
Yet another problem with microwave energy in tissue processing is that microwave energy only affects molecules with an electric dipole. Molecules with an electric dipole are known as polar, while those with little or no electric dipole are known as nonpolar. Water is a polar molecule. A water molecule has a positive charge at one end and a negative charge at the other, and is therefore twisted to and fro as it tries to align itself with the alternating electric field induced by microwaves. This molecular movement creates heat. The different concentrations of free and bound water within the specimen means that some water molecules in the tissue will be heated more than other water molecules in the tissue, thus contributing to the inconsistent heating. Microwave heating is more efficient on liquid unbound water, and much less so on fats, sugars and the like. Microwave tissue processing involves a series of events in which a tissue specimen is sequentially processed through various chemicals ranging from polar to nonpolar. As dehydration occurs and free water is removed, bound water is left behind. This bound water is a highly polarized molecule, and, therefore, is the most affected by microwave energy. Over heating of this bound water can lead to tissue distortion causing a “cooked” tissue sample. This potential for tissue distortion after dehydration is exacerbated by the fact that most traditional processing chemicals are nonpolar. Thus, excessive microwave energy is required to build up enough heat to bring about the desired chemical reactions. To minimize tissue distortion there should be a gradual change in polarity of the processing solutions from highly polar aqueous fixatives to nonpolar hardening agents.
In addition to the above referenced problems that have been encountered using microwave energy in tissue processing, there have also been attempts to use more environmentally friendly and user friendly solvents in tissue processing. Traditional solvents used in tissue processing include xylene, limonene, benzene, toluene, chloroform, and petroleum ether. These solvents are harsh in that they can damage the tissue sample. These traditional solvents are generally capable of over-hardening and shrinking tissue. Increasing the exposure to these solvents may help the processing of fatty tissue but only at the expense of delicate small biopsies.
Tissue porosity has a major impact on processing. Porosity at an ultrastructural level is determined by the nature of the tissue and the effects of processing reagents to which the tissue are subjected. Tissue porosity is a function of the number and size of natural and artificial pores, and the swelling and shrinkage of the biopolymer matrix. Even after fixation, cell surfaces continue to act as osmotic membranes. Regardless of fixative process solution used, all tissue undergoes limited shrinkage and hardening during dehydration, clearing and infiltration. Hardening generally results from tissue shrinkage, accompanied in most cases by decreased porosity. Fatty tissue specimens usually require extended processing as lipids inhibit the diffusion of processing solution. This extended processing requirement has a tendency to decrease porosity, inhibiting the removal of lipids.
These traditional solvents also present a risk to the laboratory personnel. Further, they require careful disposal so as to not damage the environment. For all of the above reasons, it would be desirable to use solvents that are not so harsh to the tissue sample, to the user, or to the environment.
It is therefore an object of the present invention to provide a rapid, efficient, and high quality tissue processing method.
It is further an object of the present invention to provide a tissue processing method that employs the benefits of ohmic heating.
It is further an object of the invention to use chemicals that provide superior processing attributes, are safer to handle, and are more environmentally friendly than current chemicals being used in tissue processing.